The major goal of the research plan is to complete the three-dimensional X-ray diffraction analysis of the E. coli protein elongation factor, Tu-GDP, and to utilize the new structural information to carry out a series of difference Fourier analyses on Tu-antibiotic complexes to elucidate the antibiotic binding site(s) on Tu. The completed structural determination at 2.8 Angstroms will result in a three-dimensional model of the protein, including the relative location of all amino acids along the polypeptide backbone and the location of the GDP binding site. The subsequent refinement of the protein structure at 2.35 Angstroms will provide an accurate description of the coordinates, conformation and interactions of all amino acids. Phase angles will be calculated from the final protein structure and applied to Fourier syntheses using, as coefficients, the difference between structure amplitudes for Tu-complexes and native Tu-GDP. A number of small molecules, including antibiotics, have successfully been diffused into crystals of Tu and diffraction data for one, a Tu-kirromycin complex, have been processed to a resolution of 2.7 Angstrom. X-ray diffraction data for other Tu-complex crystals, including the substrates, propidium iodide, tetracycline, streptomycin, puromycin, TPCK and ANS, will be collected during the grant period. An atomic description of all binding sites will be obtained by difference Fourier analyses and the results will provide a foundation for the rational design of antibiotics using Tu as a target site for the inhibition of bacterial protein biosynthesis. The refined structure of Tu will also be used as a basic model to study the atomic details of the conformational changes that occur in Tu during protein synthesis as the protein interacts with its allosteric effectors, GDP and GTP, and with other macromolecules, Ts and aminoacyl-tRNA. Large crystals of the Tu-Ts complex have been grown and a diffraction analysis will be initiated. The Tu-Ts structure will provide a molecular description of the interaction between the proteins and may suggest mechanisms for catalysis of guanine nucleotide exchange and for initiation of RNA synthesis in QB phage infection. New biochemical methods have been developed for the preparation of large quantities of the Tu-GTP-aminoacyl-tRNA complex and cocrystallization trials will resume. An X-ray diffraction analysis of the ternary complex would provide a direct visualization of a protein-nuclei acid interaction and may answer questions about Tu's ability to complex only to noninitiator, aminoacyl-tRNAs.